Western Blot
From VLP 'Lysing Cells' *Chill PBS on ice *Prepare labeled microtubes for each sample *Thaw enough urea lysis buffer to each sample (depending on amt of cells) *Get cell scraper (next to George’s bench) and paper towel sprayed with 70% EtOH *Aspirate media from cells *Rinse 2 X 1ml PBS *Add 1ml PBS and use scraper to get all cells off the dish *Transfer cells from dish to microtube and place on ice *Pulse-spin to pellet cells and place on ice *Resuspend in appropriate volume of lysis buffer by vortexing (~40-600µl depending on amount of cells) *Incubate on ice for 30 min. *Spin down to pellet cell debris *Transfer cell lysate to new microtubes 'Measuring Protein Concentration (Bradford Assay)' *Dilute BSA to generate standard curve *BSA (2mg/ml) located in 4ºC *Prepare Bradford Reagent (BR; dilute 1:5 in distilled water) *Dilute 1ul BSA in 1ml Bradford Reagent (BR) to make serial dilution *Further dilute 50ul in 1ml BR for 0.1ug 100ul in 1ml BR for 0.2ug 250ul in 1ml BR for 0.5ug 500ul in 1ml BR for 1ug *Add undiluted BSA to BR: 1ul in 1ml for 2ug 2ul in 1ml for 4ug 4ul in 1ml for 8ug 8ul in 1ml for 16ug *Prepare microtubes with 1ml BR for each sample *Add 2ul sample to each microtube with BR Incubate at rt for 5-15min *Measure A595 for standard curve and samples *Cuvettes are above Katie’s bench *Make sure the spectrophotometer has the right cuvette holder *Set the fixed wavelength to 595 *Measure the absorbance for each sample *Determine protein concentration in each sample *Normalize to lowest amount protein concentration *Divide smallest absorbance by all to get ratio of protein *Set standard amount for normalizer(i.e. use 60µl of lowest sample and adjust other sample by multiplying the ratio of lowestsample/othersamples x 60µl; add the resulting amount of lysate to a tube and dilute to 60µl with buffer) *Add Loading Dye (4X LDS from Invitrogen) 'Loading/Running PAGE' *Choose appropriate acrylamide percentage based on protein size (or use gradient gel) *10-well gels hold 25ul 12-well gels hold 20ul 15-well gels hold 15ul *For Bis-Tris gels (NuPAGE-Invitrogen, in 4C walk-in) use MOPS Running Buffer (next to Vanessa’s bench) *Remove white sticker from back of gel and set up gel apparatus *If only running one gel, use buffer dam *Add running buffer to tank (500ml per tank) *Rinse out wells by aspiration and refill with buffer *Add appropriate amount of sample to each well *Connect to power source and run at 140V for 1.5-2h; until blue dye runs out 'Semi-dry Transfer' *Prepare transfer buffer and cut Whatmann paper (blotting paper) and membrane *Rinse PVDF membrane in MeOH (do not do this with nitrocellulose membrane) *Place blotting paper and membrane in transfer buffer *Make paper(x2):gel:membrane:paper(x2) sandwich and squeeze out bubbles *Place on semi-dry transfer machine and squeeze out bubbles again *Place lid on top of machine and run at 10-12V for 1.5-2hrs *Check that current is decreasing *Once done, put in 5% milk in TBST 'Western Blot' *Blocking: Place membrane in 5% milk in TBST (5g milk in 95ml TBST; can be used for 1-2weeks after made) for one hour *Wash in TBST 3X 10 min *Put membrane in primary antibody diluted in TBST for one hour-o/n *Wash in TBST 3X 10 min *Put membrane in secondary antibody diluted in blocking solution for one hour *Wash in TBST 3X 10 min *Add ECL solutions A and B—shake vigorously for one min *Place on saran wrap and develop in dark room